Cultured Drosophila melanogaster S2 and S2R+ cell lines have become important tools for uncovering fundamental aspects of cell biology as well as for gene discovery. Despite their utility, these cell lines are nonmotile and cannot build polarized structures or cell-cell contacts. Here we outline a previously isolated, but uncharacterized, Drosophila cell line named Dm-D17-c3 (or D17). These cells spread and migrate in culture, form cell-cell junctions and are susceptible to RNA interference (RNAi). Using this protocol, we describe how investigators, upon receiving cells from the Bloomington stock center, can culture cells and prepare the necessary reagents to plate and image migrating D17 cells; they can then be used to examine intracellular dynamics or observe loss-of-function RNAi phenotypes using an in vitro scratch or wound healing assay. From first thawing frozen ampules of D17 cells, investigators can expect to begin assaying RNAi phenotypes in D17 cells within roughly 2-3 weeks.